Self-assembling replication defective hybrid virus particles

ABSTRACT

The invention pertains to self-assembled replication defective hybrid virus-like particles having capsid and membrane glycoproteins from at least two different virus types and method of making same. Recombinant viral vectors as well as the viral particles can be used as immunogens and drug delivery vehicles.

This is a divisional of copending application Ser. No. 08/017,124 filedon Feb. 12, 1993 Pat No. 5,420,026 International ApplicationPCT/US91/05650 filed on Aug. 8, 1991 and which designated the U.S. whichwas a continuation in part of U.S. application Ser. No. 07/567,828 filedAug. 15, 1990 abandoned.

BACKGROUND OF THE INVENTION

Vaccination has played a key role in the control of viral diseasesduring the past 30 years. Vaccination is based on a simple principle ofimmunity: once exposed to an infectious agent, an animal mounts animmune defense that provides lifelong protection against disease causedby the same agent. The goal of vaccination is to induce the animal tomount the defense prior to infection. Conventionally, this has beenaccomplished through the use of live attenuated or whole inactivatedforms of the virus as immunogens. The success of these approachesdepends on the presentation of native antigen which elicits the completerange of immune responses obtained in natural infection.

Despite their considerable success, conventional vaccine methodologiesare subject to a number of potential limitations. Insufficientlyinactivated vaccines may cause the disease they are designed to prevent.Attenuated strains can mutate to become more virulent ornon-immunogenic. Viruses that can establish latency, such as theherpesviruses, are of particular concern as it is not known whetherthere are any long-term negative consequences of latent infection byattenuated strains. Finally, there are no efficient means of growingmany types of viruses.

Recent advances in recombinant DNA technology offer the potential fordeveloping vaccines based on the use of defined antigens as immunogens,rather than the intact infectious agent. These include peptide vaccines,consisting of chemically synthesized, immunoreactive epitopes; subunitvaccines, produced by expression of viral proteins in recombinantheterologous cells; and the use of live viral vectors for thepresentation of one or more defined antigens.

Both peptide and subunit vaccines are subject to a number of potentiallimitations. A major problem is the difficulty of ensuring that theconformation of the engineered proteins mimics that of the antigens intheir natural environment. Suitable adjuvants and, in the case ofpeptides, carrier proteins, must be used to boost the immune response.In addition these vaccines elicit primarily humoral responses, and thusmay fail to evoke effective immunity. Subunit vaccines are oftenineffective for diseases in which whole inactivated virus can bedemonstrated to provide protection. For example, canine parvovirussubunits fail to elicit virus-neutralizing antibodies in rabbits (Smithand Halling, Gene, 29:263-269 (1984)), although protective inactivatedvaccines are available.

As an alternative to recombinant-produced subunit vaccines comprising apurified polypeptide, it may be possible to develop non-infectious,subunit-like vaccines that consist of viral capsid proteins assembledinto virus-like structures. Such non-replicating, virus-like particleswould have many of the immunologic advantages of inactivated vaccinescombined with the safety features of subunit vaccines. Severalresearchers have reported the development of eukaryotic systems for theexpression of foreign viral capsid proteins, and the self assembly ofthese proteins into virus-like particles. For example, co-expression ofcanine parvovirus (CPV) capsid proteins VP1 and VP2 in murine cellstransformed with a bovine papilloma virus/CPV recombinant plasmidresulted in the formation of self-assembling particles that resembled,biochemically and immunologically, authentic CPV virions (Mazzara, etal., Modern Approaches to Vaccine, Cold Spring Harbor Laboratory, N.Y.,R. M. Chanock and R. A. Lerner, eds. pp. 419-424 (1986); Mazzara, etal., PCT Application No. WO88/02026, published Mar. 24, 1988). When usedto vaccinate susceptible dogs, these empty capsids elicited immuneresponses capable of protecting against CPV challenge. In anotherexample, it has been shown that the expression of HIV or SIV gagprecursor polypeptide in insect cells using the baculovirus expressionsystem results in the formation of immature, retroviral-like particlesthat are secreted into the culture medium of infected cells (Gheysen, etal., Cell, 59:103-112 (1989); Delchambre, et al., EMBO J., 8:2653-2660(1989)). In mammalian cells, HIV-like particles that contained corepolypeptides as well as reverse transcriptase were produced aftertransient expression of the HIV gag-pol genes using an SV40 latereplacement vector (Smith, et al., J. Virol., 64:2653-2659 (1990)).

Recombinant vaccinia viruses that express at least the HIV gag gene havealso been shown to give rise to the production of retroviral-likeparticles upon infection of appropriate host cells (Karacostas, et al.,Proc. Natl. Acad. Sci. USA, 86:8964-8967 (1989); Shiota and Shibuta,Virology, 175:139-148 (1990)). The coexpression in recombinantvaccinia-infected cells of gag polypeptides with the HIV envelopeglycoproteins resulted in the formation of HIV-like particles thatcomprised an enveloped core structure containing, embedded in theenvelope, the HIV envelope glycoproteins. The coexpression of gag andenv genes in infected cells could be achieved by co-infecting the cellswith two different recombinant vaccinia viruses, one expressing env andone expressing gag-pol (Haffar, et al., J. Virol., 64:2653-2659 (1990)),or by infecting the cells with a single recombinant that expressed bothenv and gag-pol (Mazzara, et al., U.S. patent application Ser. Nos.07/360,027 and 07/540,109).

The ability to produce particles containing viral envelope glycoproteinshas important implications for vaccine development. Viral envelopeglycoproteins, which are located in the outer lipid membrane ofenveloped viruses (such as herpesviruses, retroviruses, togaviruses,rhabdoviruses, paramyxoviruses, orthomyxoviruses and coronaviruses) areoften the major immunogenic determinants of the virus. In the case ofHIV, for example, the envelope glycoprotein gp120 contains the keyepitopes that elicit virus-neutralizing antibody responses (Arthur, L.A., et al., Proc. Natl. Acad. Sci. USA, 84:8583-8587 (1987)). Similarly,the herpes simplex virus glycoprotein gB and the rabies glycoproteinboth elicit virus-neutralizing antibody responses and, in addition, havebeen shown to protect against challenge with the cognate pathogens inthe absence of other viral proteins (Paoletti, et al., Proc. Natl. Acad.Sci. USA, 81:193-197 (1984); Wiktor, et al., Proc. Natl. Acad. Sci. USA,81:7194-7198 (1984)).

Unfortunately, there are many viruses for which heterologous expressionof self-assembling viral capsids may not prove feasible. Formation ofherpesviruses capsids, for example, would require the expression of moregenes than can be practically accommodated in available expressionvectors. The mechanism of particle assembly for a number of otherviruses, such as the helical RNA viruses, makes self assembly ofvirus-like particles from a heterologous expression system problematic.Nonetheless, it would be useful to be able to produce non-infectious,self-assembling virus-like particles containing membrane glycoproteinsfrom any enveloped virus.

Envelope glycoproteins from viruses of different families can beincorporated at low frequency into heterologous virus particles by thebiological phenomenon known as pseudotyping or phenotypic mixing. Inco-infection experiments, the genome of one virus species can bedemonstrated to be physically associated with glycoproteins from theother species. In a review of the literature on this phenomenon, Zavada,(J. Gen. Virol., 63:15-24 (1982)) cites examples of pseudotypingbetween, for example, retroviruses and togaviruses, rhabdoviruses,paramyxoviruses or herpesviruses. For pseudotyping to occur, the twoviruses must have compatible life cycles, i.e., neither must interferewith the replication of the other. Recently, Zhu, et al., (J. AcquiredImmune Deficiency Syndromes, 3:215-219 (1990)) described phenotypicmixing between HIV and vesicular stomatitis virus or herpes simplexvirus.

SUMMARY OF THE INVENTION

This invention pertains to self-assembling, replication defective,hybrid virus-like particles. These particles, which contain polypeptidesor portions of polypeptides from at least two different viral species,comprise assembled capsid polypeptides from one virus species surroundedby a membrane containing at least a portion of one or more viralenvelope glycoproteins from one or more different virus species. Theparticles are produced using recombinant DNA viruses that express: (1)heterologous genes encoding virus capsid proteins and (2) a homologousor heterologous gene encoding an envelope glycoprotein. The capsidproteins and the envelope glycoprotein may be encoded in the samerecombinant virus; in this case, infection of suitable host cells withthe recombinant virus will result in the production of hybrid virus-likeparticles containing the encoded heterologous capsid proteins and theenvelope glycoprotein. Alternatively, the capsid proteins and theenvelope glycoprotein may be encoded in two or more different carrierviruses of the same species. In this case, hybrid virus-like particlesare produced by co-infection of suitable host cells with the carriervirus.

This invention also pertains to the recombinant viruses expressing theproteins that comprise the particle and to the intermediate DNA vectorsthat recombine with the parent virus in vivo or in vitro to produce therecombinant virus. In addition, this invention pertains to methods ofproducing non-replicating, self-assembling hybrid virus particles andmethods of using the particles as a biopharmaceutical in an appropriateformulation or using the recombinant virus expressing the particles as adelivery vehicle.

The hybrid virus-like particles and/or the virus capable of expressingthe particles can be used as a vaccine against the correlateheterologous pathogens. The particles may contain, for example, capsidpolypeptides from retroviruses (such as HIV, SIV, felineimmunodeficiency virus (FIV), murine retroviruses, equine infectiousanemia, visna virus and other retroviruses) or from other envelopedviruses in association with envelope glycoproteins from herpesviruses,retroviruses, togaviruses, rhabdoviruses, paramyxoviruses,orthomyxoviruses or coronaviruses. These particles can be used alone asimmunogens or used in combination with other immunogens for vaccinationagainst pathogenic viruses or for therapeutic purposes such as enhancingimmune responses in an infected individual. The hybrid virus-likeparticles of this invention can also be used for targeted delivery oftherapeutic agents, such as cytotoxic drugs or nucleic acids to specificcell types.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the construction of plasmid pAbT4660 which contains theentire SIV gag-pol region under the transcriptional control of thevaccinia 40K promoter.

FIG. 2 shows the construction of plasmid pAbT4602 which contains thepseudorabies virus gIII gene under the transcriptional control of thevaccinia 40K promoter.

FIG. 3 shows the construction of plasmid pAbT1527 which contains the gDgene of Herpes Simplex Virus Type 2.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to self-assembling, replication defective,hybrid virus-like particles. The virus particles are hybrids in thatthey contain polypeptides from at least two different viral species. Theinvention is designed to take advantage of the phenomenon ofpseudotyping to achieve assembly of the heterologous polypeptides intonovel hybrid virus particles. The phenomenon of pseudotyping has beenpreviously described in a review by Zavada, J. Gen. Virol., 63:15-24(1982).

Preferably, hybrid virus-like particles will contain retroviral capsidpolypeptides, e.g., capsid polypeptides from lentiviruses, such as HIV,SIV, FIV, equine infectious anemia, visna virus, or from otherretroviruses such as murine leukemia virus. The particles will alsocontain envelope glycoproteins from a different viral species. Suchother viruses can be DNA or RNA viruses. The particles may furthercontain other desirable polypeptides appropriately linked to a viralenvelope glycoprotein. The viral particles can have substantially littleor no RNA packaged within the particle; or they can contain specific RNAfor delivery of heterologous genes to a targeted cell. Methods forproducing such viral particles have been described in U.S. applicationSer. No. 07/540,109, filed Jun. 19, 1990, the teachings of which areincorporated herein by reference.

The method of producing hybrid virus-like particles, recombinant virusesexpressing these particles, and uses therefor will be discussed indetail below and in the Examples section.

1. Genes Encoding Viral Capsid Polypeptides

Genes encoding viral polypeptides capable of self assembly intodefective, nonself-propagating hybrid viral particles can be obtainedfrom the genomic DNA of a DNA virus or the genomic cDNA of an RNA virusor from available subgenomic clones containing the genes. These geneswill include those encoding viral capsid proteins (i.e., proteins thatcomprise the viral protein shell). Additional viral genes may also berequired for capsid protein maturation and particle self-assembly. Thesemay encode, for example, viral proteases responsible for processing ofcapsid protein.

One virus genus from which genes encoding self-assembling capsidproteins can be isolated is the lentiviruses, of which HIV is anexample. The HIV gag protein is synthesized as a precursor polypeptidethat is subsequently processed, by a viral protease, into the maturecapsid polypeptides. However, the gag precursor polypeptide canself-assemble into virus-like particles in the absence of proteinprocessing. Gheysen, et al., Cell, 59:103 (1989); Delchambre, et al.,The EMBO J., 8:2653-2660 (1989). HIV capsids are surrounded by a loosemembranous envelope that contains the viral glycoproteins. In the nativevirus these are encoded by the HIV env gene.

2. Envelope Proteins

In order to create hybrid, non-self-propagating particles, part or allof the gene(s) for one or more envelope glycoproteins from a virus otherthan that used as the source of capsid genes are required.

Genes encoding envelope glycoproteins can be isolated from any of alarge number of diverse, enveloped viruses. These viruses can be ofeither the DNA or RNA classes. Examples of enveloped viruses includeherpesviruses, retroviruses, togaviruses, rhabdoviruses,paramyxoviruses, orthomyxoviruses and coronaviruses. Envelopeglycoproteins are typically characterized by distinct intracellular,extracellular and transmembrane regions. An enveloped virus may expressone or more envelope glycoproteins, which are often the majorimmunogenic determinants of the virus. Viral envelope glycoproteins mayalso be responsible for targeting specific cell surface receptors forvirus adsorption and penetration into cells.

3. Parent Viruses

A number of viruses, including retroviruses for example, HIV, SIV, FIV,equine infectious anemia, and visna virus, adenoviruses, herpesvirusesand pox viruses, have been developed as live viral vectors for theexpression of heterologous antigens. Cepko, et al., Cell, 37:1053-1062(1984); Morin, et al., Proc. Natl. Acad. Sci. USA, 84:4626-4630 (1987);Lowe, et al., Proc. Natl. Acad. Sci. USA, 84:3896-3900 (1987); Panicali& Paoletti, Proc. Natl. Acad. Sci. USA, 79:4927-4931 (1982); Mackett, etal., Proc. Natl. Acad. Sci. USA, 79:7415-7419 (1982). The examples givenillustrate the use of the pox virus family. The preferred pox virus isvaccinia virus, a relatively benign virus which has been used for yearsas a vaccine against smallpox. Vaccinia virus has been developed as aninfectious eukaryotic cloning vector (Paoletti and Panicali, U.S. Pat.No. 4,603,112) and recombinant vaccinia virus has been used successfullyas a vaccine in several experimental systems. The virus is considerednononcogenic, has a well-characterized genome, and can carry largeamounts of foreign DNA without loss of infectivity. Mackett, M. and G.L. Smith, J. Gen. Virol., 67:2067 (1986). Another preferred pox virus isfowl pox virus, a pathogen of poultry. This virus has also beendeveloped into a eukaryotic cloning vector. Boyle, et al., PCTApplications WO88/02022 published Sep. 22, 1987 and WO89/07644 publishedAug. 24, 1989; Yanagida, et al., EP284416 published Sep. 28, 1988; PCTApplication WO90/02191, published Mar. 8, 1991.

4. DNA Vectors for In Vivo Recombination with a Parent Virus

According to the method of this invention, viral genes that code forpolypeptides capable of assembly into replication defective, hybridviral particles are inserted into the genome of at least one parentvirus in such a manner as to allow them to be expressed by that virusalong with the expression of the normal complement of parent virusproteins. This can be accomplished by first constructing a DNA donorvector for in vivo recombination with a parent virus.

In general, the DNA donor vector contains the following elements:

(a) a prokaryotic origin of replication, so that the vector can beamplified in a prokaryotic host;

(b) a gene encoding a marker which allows selection of prokaryotic hostcells that contain the vector (e.g., a gene encoding antibioticresistance);

(c) heterologous genes from at least two different viruses each genelocated adjacent to a transcriptional promoter (e.g., the vaccinia 7.5K,30K, 40K, 11K or BamF promoters or modified versions of these promoters)capable of directing the expression of adjacent genes; and

(d) DNA sequences homologous to the region of the parent virus genomewhere the foreign gene(s) will be inserted, flanking the construct ofelement c (e.g., the vaccinia TK or HindIII M sequences).

Methods for constructing donor plasmid for the introduction of multipleforeign genes into pox virus are described in EP 0261940, published Mar.30, 1988, entitled "Pseudorabies Vaccine," the techniques of which areincorporated herein by reference. In general, all viral DNA fragmentsfor construction of the donor vector, including fragments containingtranscriptional promoters and fragments containing sequences homologousto the region of the parent virus genome into which foreign genes are tobe inserted, can be obtained from genomic DNA or cloned DNA fragments.

The donor vector preferably contains an additional gene which encodes aselectable marker under control of a separate promoter which will allowidentification of recombinant viruses containing inserted foreign DNA.Several types of marker genes can be used to permit the identificationand isolation of recombinant viruses. These include genes that encodeantibiotic or chemical resistance (e.g., see Spyropoulos, et al., J.Virol., 62:1046 (1988); Falkner and Moss, J. Virol., 62:1849 (1988);Franke, et al., Mol. Cell. Biol., 5:1918 (1985)), as well as genes, suchas the E. coli lacZ gene, that permit identification of recombinantviral plaques by calorimetric assay. (Panicali, et al., Gene, 47:193-199(1986)).

A method for the selection of recombinant vaccinia viruses relies upon asingle vaccinia-encoded function, namely the 29K host-range geneproduct. Gillard, et al., Proc. Natl. Acad. Sci. USA, 83:5573 (1986).This method was described in PCT Application No. WO89/12103, publishedDec. 18, 1989, entitled "Methods of Selecting for Recombinant PoxViruses," the teachings of which are incorporated herein by reference.

5. Integration of Foreign DNA Sequences into the Viral Genome andIsolation of Recombinants

Homologous recombination between donor plasmid DNA and viral DNA in aninfected cell results in the formation of recombinant viruses thatincorporate the desired elements. Appropriate host cells for in vivorecombination are generally eukaryotic cells that can be infected by thevirus and transfected by the plasmid vector. Examples of such cellssuitable for use with a pox virus are chick embryo fibroblasts, RK13(rabbit) cells, HuTK143 (human) cells, and CV-1 and BSC-40 (both monkeykidney) cells. Infection of cells with pox virus and transfection ofthese cells with plasmid vectors is accomplished by techniques standardin the art (Panicali and Paoletti, U.S. Pat. No. 4,603,112).

Following in vivo recombination, recombinant viral progeny can beidentified by one of several techniques. For example, if the DNA donorvector is designed to insert foreign genes into the parent virusthymidine kinase (TK) gene, viruses containing integrated DNA will beTK⁻ and can be selected on this basis (Mackett, et al., Proc. Natl.Acad. Sci. USA, 79:7415 (1982)). Alternatively, co-integration of a geneencoding a marker or indicator gene with the foreign gene(s) ofinterest, as described above, can be used to identify recombinantprogeny. One preferred indicator gene is the E. coli lacZ gene:recombinant viruses expressing β-galactosidase can be selected usingchromogenic substrate for the enzyme (Panicali, et al., Gene, 47:193(1986)). A second preferred indicator gene for use with recombinantvaccinia virus is the vaccinia 29K gene: recombinant viruses thatexpress the wild type 29K gene-encoded function can be selected bygrowth on RK-13 cells. Another method by which recombinant virusescontaining genes of interest can be identified is by an in situ enzymebased immunoassay performed on virus plaques which detects foreignprotein expressed by vaccinia-infected cells.

As described more fully in the Examples, donor plasmids containing SIVor pseudorabies virus genes could be recombined into vaccinia viruseseither at the HindIII M region or TK region. Using either insertionsite, recombinant viruses can be selected as described above.

6. Characterizating the Viral Antigens Expressed by Recombinant Viruses

Once a recombinant virus has been identified, a variety of methods canbe used to assay the expression of the polypeptide encoded by theinserted gene. These methods include black plaque assay (an in situenzyme immunoassay performed on viral plaques), Western blot analysis,radioimmunoprecipitation (RIPA), and enzyme immunoassay (EIA).Antibodies to antigens expressed by viral pathogens are either readilyavailable, or may be made according to methods known in the art. Forexample, for simian immunodeficiency virus, the antibodies can be serafrom macaques infected with SIV.

7. Viral Particle Formation

Expression analysis described in the preceding section can be used toconfirm the synthesis of the polypeptides encoded by insertedheterologous viral genes, but does not address the question of whetherthese polypeptides self-assemble, in vivo or in vitro, into replicationdefective viral particles. This can readily be determined empiricallybased upon the present disclosure.

Cells can be infected in vitro with one DNA carrier virus expressing acapsid polypeptide, for example, retroviral gag or gag-pol genes, and asecond carrier virus expressing an envelope glycoprotein gene.Preferably, the cell is co-infected. More preferably, it is co-infectedwith the same carrier DNA virus. Alternatively, a single carrier virusthat expresses both a capsid polypeptide gene and an envelope gene canbe used.

For self assembly to occur, the capsid and env gene products need to beexpressed at about the same time. This can readily be accomplished by avariety of methods well known to the person of ordinary skill in theart. For example, one can use a viral vector containing the heterologousenv and capsid genes. Alternatively, one can co-infect a cell with twoviral vectors where one expresses the heterologous capsid genes and asecond viral vector containing a gene expressing an env glycoprotein.Preferably, the viral vectors would have a similar life cycle so thatthe capsid and env gene products are expressed at about the same time.Still more preferably, the viral vectors would correspond to the sameviral genome. In another embodiment, one can have the env and or capsidgene under the control of an inducible promoter, see for example,Haynes, et al., PCT Application No. WO91/05865, published May 2, 1991.Thus, one can turn these genes "on" at about the same time, so that onecan obtain the expression of their gene products at about the same time,thereby resulting in self-assembly of the particle. In anotherembodiment, only one of the genes needs to be under the control of aninducible promoter, for example, the human metallothionein IIa promoter.One can then transform a cell containing this viral gene with the otherviral vector, induce the gene already in the cell to express the capsidgene or the env gene under its control so that its expression coincideswith that of the gene on the vector being used to transform the cell.

In order to characterize the defective hybrid viral particles producedby recombinant viruses expressing heterologous viral polypeptides, cellscan be infected with the recombinant virus(es) in the presence ofradiolabeled amino acid. High speed centrifugation can then be used tosediment particles from the culture medium. The pellet resulting fromcentrifugation of the culture medium can be resuspended and both thepellet and the supernatant can be immunoprecipitated with appropriateantisera to analyze the polypeptides present in each fraction. Forexample, in the case of recombinants expressing SIV capsid polypeptides,macaque anti-SIV antisera can be used for the analysis of capsidpolypeptides. A second antibody, specific for the glycoprotein, would beused to detect the presence of the glycoprotein in the particlepreparation.

To further characterize the material in the pellet resulting fromcentrifugation of the culture medium, the pellet can be resuspended andanalyzed by centrifugation through a sucrose density gradient. Thegradient can then be fractionated and the fractions immunoprecipitatedwith the appropriate antisera. These experiments show whether the pelletcontains capsid material banding at the density expected for defectiveviral particles, and whether the envelope glycoprotein is specificallyassociated with the defective viral particles banding at this density.

Alternatively, formation of hybrid particles can be demonstrated usingelectron microscopy. After infection of appropriate host cells with therecombinant virus(es) expressing capsid and envelope glycoprotein genes,particles can be harvested from the culture medium by high speedcentrifugation as described above. The presence of envelopeglycoproteins on the surface of the particles can be demonstrated byimmunogold staining, using a monoclonal antibody directed against theenvelope glycoprotein, followed by electron microscopic examination.

8. Vaccines

Live recombinant viral vectors that express heterologous viral antigenscapable of self-assembly into replication defective hybrid virusparticles can be used to vaccinate humans or animals susceptible toinfection if the viral vector used to express the heterologous defectivevirus particles infects but does not cause significant disease in thevaccinated host. Examples of such benign viral vectors include certainpox viruses, adenoviruses, and herpesviruses.

Alternatively, the defective hybrid virus particles produced by theserecombinant vector viruses can be isolated from the culture medium ofcells infected in vitro with the recombinant vector viruses. Thepurified particles used for vaccination of individuals susceptible toviral infection will authentically present envelope glycoproteins to thehost immune system, but will not contain infectious viral geneticmaterial. Consequently, they offer the advantage of conventional killedvirus vaccine preparations, yet circumvent the major drawbacks to theuse of killed virus as a vaccine for the prevention of infection. Theseinclude the danger of incomplete inactivation of killed viruspreparations and, in the case of certain viruses, such as retroviruses,the reluctance to introduce a complete viral genome (the HIV genome, forexample) into seronegative individuals.

Vaccine compositions utilizing these replication defective hybrid virusparticles would generally comprise an immunizing amount of the viralparticles in a pharmaceutically acceptable vehicle. The vaccines wouldbe administered in a manner compatible with the dosage formulation, andin such amount as would be therapeutically effective and immunogenic.

Finally, the purified particles may be used in combination with liverecombinant viruses as part of a total vaccination protocol, either asthe primary immunizing agent, to be followed by vaccination with liverecombinant virus, or to boost the total immune response after primaryvaccination with live recombinant virus.

9. Therapeutic Use of Recombinant Viruses Expressing Viral AntigensCapable of Assembling into Defective Hybrid Viral Particles: TherapeuticUse of Defective Hybrid Viral Particles Produced by These RecombinantViruses

Even if immunization can not protect against initial infection,immunization of a previously infected individual with the hybridparticles might, for certain viruses, sufficiently boost immunity toprotect against the onset of disease. This is, for example, how rabiesvaccine is used therapeutically. Alternatively, for viruses thatestablish latency, immunization of an infected individual might prolongthe latency period of that virus within the individual. (Salk, Nature,327:473-476 (1987)). This may be particularly important in the case ofviral infections characterized by long latency periods, such as HIV orherpesvirus infections.

The defective hybrid viral particles of this invention can also be usedto deliver heterologous genes (e.g., antisense genes, genes encodingtoxins, genes encoding an immunogen) to a targeted cell. Methods forproducing such viral particles have been described in U.S. patentapplication Ser. No. 07/540,109, filed Jun. 19, 1990, the teachings ofwhich are incorporated herein by reference. Hybrid viral particles couldbe used to deliver mRNAs that are directly translated in the target cellinto the encoded protein product. Alternatively, specific RNA packagedwithin hybrid retroviral particles that contain active reversetranscriptase and other pol-encoded functions could be delivered to thetargeted cells and reverse transcribed into DNA. This DNA could thenintegrate into the host genome, and the encoded genes would be expressedby host transcription/translation machinery. These approaches could beused to deliver genes encoding products toxic to the targeted cells(e.g., virally infected cells). In another application, particlescontaining RNA encoding heterologous genes could be administered to anindividual in order to elicit immune responses to the encoded geneproducts.

10. Therapeutic Use of Defective Hybrid Virus Particles as Agents forTargeted Drug Delivery

Defective, nonself-propagating virus particles can also be used todeliver certain drugs (e.g., cytotoxic drugs, antiviral agents, nucleicacids) to virus receptor-bearing cells. Such drugs may be coupled, bytechniques known in the art, to the outer surface of the virus particle,or incorporated within, and delivered with high specificity to targetcells. For example, cytotoxic drugs may be coupled to defective HIVparticles and delivered with a high degree of specificity to CD4⁺ Tcells, since the HIV envelope glycoprotein present on these particlesbind specifically and with high affinity to the CD4 molecule.

Specific targeting of therapeutic agents can be achieved by selecting asthe heterologous glycoprotein one with a tropism for surface receptorson specific cell types. For example, hybrid particles containingherpesvirus glycoproteins might be used to target cells of the nervoussystem, whereas hybrid particles containing the hepatitis B surfaceantigen would target hepatic cells.

The invention will be further illustrated by the following examples:

EXAMPLES General Procedures

Cells and Virus

E. coli strain MC1061 (Casadaban and Cohen, J. Mol. Biol., 138:179(1980)) was used as the host for the growth of all plasmids. The monkeykidney cell line BSC-40 (Brockman and Nathans, Proc. Natl. Acad. Sci.USA, 71:942 (1974)) and the rabbit kidney cell line RK-13 (ATCC No.CCL37; Beale, et al., Lancet, 2:640 (1963)) were used for vacciniainfections and transfections. Cells were propagated in Dulbecco modifiedEagles Medium (DME, Gibco, Grand Island, N.Y.) supplemented with 5%fetal calf serum (FCS).

A 29K- lacZ+ strain vAbT33 (see U.S. patent application Ser. No.205,189, filed Jun. 10, 1988, the teachings of which are incorporatedherein by reference) was used as the parental virus for in vivorecombination. Viral infection, transfections, plaque purification andvirus amplification were performed essentially as described(Spyropoulos, et al., J. Virol., 62:1046 (1988)).

Molecular Cloning Procedures

Restriction enzyme digestions, purification of DNA fragments andplasmids, treatment of DNA with Klenow, T4 DNA polymerase, calfintestinal alkaline phosphatase, T4 DNA ligase, or linkers andtransformation of E. coli were performed essentially as described(Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1982, the teachings ofwhich are incorporated herein by reference). Restriction enzymes wereobtained from New England Biolabs or Boehringer-Mannheim. The largefragment of DNA polymerase (Klenow) was obtained from United StatesBiochemical Corporation, T4 DNA polymerase was obtained from New EnglandBiolabs, and T4 DNA ligase and calf intestinal alkaline phosphatase wereobtained from Boehringer-Mannheim.

EXAMPLE 1 Construction of Recombinant Plasmids Containing the gag-polRegion of Simian Immunodeficiency Virus (SIV)

This example illustrates the construction of a recombinant plasmidcontaining SIV genes for in vivo recombination with vaccinia virus (IVRvector). The construction and structure of plasmids pAbT4579 isdescribed in PCT Application No. WO89/12095, published Dec. 14, 1989.The construction and structure of plasmids pAbT4592 and pAbT4593 aredescribed in U.S. patent application Ser. No. 360,027 filed Jun. 1,1989. The teachings of these Applications are incorporated herein byreference.

a. Construction of pAbT4660 (FIG. 1).

Plasmid pAbT4592 was partially digested with HindIII, then digested tocompletion with SacI. An approximately 4990 base pair (bp) fragmentcontaining a bacterial replicon, vaccinia sequences for integration intothe HindIII M region of the genome, the vaccinia 40K promoter and theSIV gag gene was isolated. This fragment was ligated to a 3780 bpfragment resulting after digestion of plasmid pAbT4579 to completionwith HindIII and SacI, to yield plasmid pAbT4660. pAbT4660 contains theentire SIV gag-pol region under the transcriptional direction of thevaccinia 40K promoter.

EXAMPLE 2 Construction of a Recombinant Plasmid Containing the gIII Geneof Pseudorabies Virus (PRV)

This example illustrates the construction of a recombinant plasmidcontaining the PRV gIII gene for in vivo recombination with vacciniavirus (IVR vector). The construction and structure of plasmid pAbT175 isdescribed in EP 0261940, published Mar. 30, 1988. The construction andstructure of plasmid pAbT4587 is described in PCT Application No.WO90/01546, published Feb. 22, 1990. The teachings of these Applicationsare incorporated herein by reference.

a. Construction of pAbT4602 (FIG. 2).

Plasmid pAbT175 was digested to isolate a 2500 bp NcoI fragmentcontaining PRV gIII gene. The ends were repaired with the Klenowfragment of DNA polymerase I. This was ligated to vector pAbT4587 whichhad been digested with SmaI and treated with calf intestinalphosphatase. This situated the gIII gene downstream of the vacciniavirus 40K promoter to generate pAbT4602.

EXAMPLE 3 Construction of Recombinant Vaccinia Viruses Containing theSIV gag-pol Region or the PRV gIII Gene

In vivo recombination is a method whereby recombinant vaccinia virusesare created (Nakano, et al., Proc. Natl. Acad. Sci. USA, 79:1593 (1982);Paoletti and Panicali, U.S. Pat. No. 4,603,112). These recombinantviruses are formed by transfecting DNA containing a gene of interestinto cells which have been infected by vaccinia virus. A small percentof the progeny virus will contain the gene of interest integrated into aspecific site on the vaccinia genome. These recombinant viruses canexpress genes of foreign origin. Panicali and Paoletti, Proc. Natl.Acad. Sci. USA, 79:4927 (1982); Panicali, et al., Proc. Natl. Acad. Sci.USA, 80:5364 (1983).

a. Insertion of SIV gag-pol Genes into Vaccinia Strain vAbT33

To insert SIV gag-pol genes into the vaccinia virus genome at theHindIII M region of vaccinia virus strain vAbT33, a selection schemebased upon the 29K host-range gene, which is located in this region, wasused. Gillard, et al., Proc. Natl. Acad. Sci. USA, 83:5573 (1986).Recombinant vaccinia virus vAbT33 contains the lacZ gene in place of aportion of the 29K gene. This lacZ insertion destroys the function ofthe 29K gene; therefore, vAbT33 grows poorly on RK-13 cells, whichrequire the 29K gene product. Furthermore, vAbT33 forms blue plaques onpermissive cells in the presence of the chromogenic substrate forβ-galactosidase, Bluogal™, due to the presence of the lacZ gene. See PCTApplication No. WO89/12103, published Dec. 18, 1989.

IVR vector pAbT4660 was transfected into BSC-40 cells which had beeninfected with vaccinia virus vAbT33. Viral infection and plasmidtransfection were performed essentially as described. Spyropoulos, etal., J. Virol., 62:1046 (1988). Recombinant viruses were selected aswhite plaques in the presence of Bluogal™ on RK-13 cells. Plaques werepicked and purified, and the final recombinant, designated vAbT394, wasamplified.

b. Insertion of the PRV gIII Gene into Vaccinia Strain vAbT33.

To insert the PRV gIII gene into the vaccinia virus genome at theHindIII site of vaccinia virus, BSC-40 cells were infected with vAbT33,transfected with pAbT4602 and the recombinant selected by the schemedescribed in Example 3a. This generated vaccinia recombinant vAbT282.

c. Southern Blot Analysis of vAbT394 and vAbT282.

DNA was extracted from vaccinia virus-infected cells as describedEsposito, et al., J. Virol. Methods, 2:175 (1981)! and analyzed byrestriction enzyme digestions and Southern hybridization withradiolabeled probes corresponding to the SIV gag-pol genes or PRV gIIIgene as described. Maniatis, et al., Molecular Cloning: LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).This analysis confirmed the presence of these SIV and PRV sequences inthe recombinant viruses.

EXAMPLE 4 Immunoprecipitation of SIV and PRV Antigens from CellsInfected with Recombinant Vaccinia Viruses

Metabolic labeling with ³⁵ S!-methionine of BSC-40 or RK-13 cellsinfected with either recombinant vaccinia viruses vAbT394 and vAbT282and subsequent immunoprecipitation analyses were performed essentiallyas described in EP 0261940, published Mar. 30, 1988, the teachings ofwhich are incorporated herein by reference. A monoclonal antibodydesignated M7 (Hampel, et al., J. Virol., 52:583-590 (1984)) was usedfor immunoprecipitation of gIII expressed by vAbT282; IgG purifiedmacaque anti-SIV antiserum was used for immunoprecipitation of the SIVproteins expressed by vAbT394. The results, which are summarized inTable 1, show that each of these vaccinia recombinants expresses theencoded polypeptide(s).

                  TABLE 1    ______________________________________    Immunoprecipitation of SIV and PRV polypeptides    from recombinant vaccinia viruses    Vaccinia     Inserted    Proteins    recombinants genes       Observed    ______________________________________    vAbT394      SIV gag-pol p66, p55, p42, p32,                             p27, p17, p10, p9    vAbT292      PRV gIII    gp76    ______________________________________

EXAMPLE 5 Detection of Hybrid Retroviral Particles Produced byCoinfection with Vaccinia Recombinants vAbT282 and vAbT394

To demonstrate that vaccinia recombinant vAbT394 (SIV gag-pol) producesretroviral-like particles upon infection of mammalian cells, and to showthat coinfection of mammalian cells with vAbT394 and vAbT282 (PRV gIII)results in the production of hybrid retroviral-like particles containingSIV core proteins and PRV gIII envelope glycoprotein, the followingexperiment was performed: BSC-40 cells were infected with vAbT394 andvAbT282 individually and in combination, in the presence of ³⁵S!-methionine as described in Example 3. After 16-18 hours of infection,the culture medium from each infection was collected and clarified bycentrifugation twice at 3000 rpm for 5 minutes. The clarified media werethen centrifuged at 25,000 rpm for 90 minutes. After removal of thesupernatants, the resulting pellets were each resuspended in 400 μl ofIP buffer (10 mM Tris pH 7.2, 0.5 mM NaCl, 1% Triton X-100, 1% NaDOC,0.1% SDS, 5 mM EDTA, 100 mM PMSF, 10 mg/ml soybean trypsin inhibitor).Each of the three pellet samples were then subjected toimmunoprecipitation analysis using macaque anti-SIV antibody andanti-PRV gIII monoclonal antibody M7 as described in Example 4. Theresults are shown in Table 2.

                  TABLE 2    ______________________________________    Immunoprecipitation of SIV and PRV polypeptides from virus-like    particles released into the medium of cells infected with    vAbT394 and/or vAbT282    Infecting       Antibody     Proteins    virus(es)       used         Observed    ______________________________________    vAbT394 (SIV gag-pol)                    M7           none    vAbT394         macaque anti-SIV                                 p66, p55, p42,                                 p32, p27, p17,                                 p10, p9    vAbT282 (PRV gIII)                    M7           gp76 (weak)    vAbT282         macaque anti-SIV                                 none    vAbT394 + vAbT282                    M7           gp76    vAbT394 + vAbT282                    macaque anti-SIV                                 p66, p55, p42,                                 p32, p27, p17,                                 p10, p9    ______________________________________

These results showed that vAbT394 produces structures containing gag andpol polypeptides that are released into the culture medium of infectedcells and can be pelleted from the medium by high-speed centrifugation.These structures are likely to be retroviral-like particles.Additionally, coinfection of cells with vAbT394 and vAbT282 results inthe production of extracellular structures that contain both SIVpolypeptides and PRV gIII. The amount of gIII detected byimmunoprecipitation of material pelleted from culture media wasconsiderably higher when cells are coinfected with vAbT394 and vAbT282than when cells were infected with vAbT282 alone. These resultssuggested that coinfection with the two recombinant vaccinia virusesresulted in the production of hybrid virus-like particles comprising anSIV core surrounded by a membrane containing PRV gIII glycoproteinmolecules.

EXAMPLE 6 Detection of Hybrid Retroviral Particles Produced byCoinfection with Vaccinia Recombinants that Express SIV and PRV AntigensUsing Sucrose Gradient Sedimentation and Radioimmuno-Precipitation

To confirm the production of retroviral-like particles containing bothSIV capsid polypeptides and PRV gIII glycoproteins from cells coinfectedwith vAbT282 and vAbT394, the following experiments were performed.

BSC-40 cells were coinfected with the recombinant vaccinia virusesvAbT282 and vAbT394 at a multiplicity of 10 plaque-forming units (pfu)of each recombinant per cell in the presence of ³⁵ S!-methionine asdescribed in Example 4. After 20-24 hours of infection, the culturemedium was collected and clarified by centrifugation twice at 3000 rpmfor 5 minutes. The clarified medium was then centrifuged at 25,000 rpmfor 90 minutes to pellet the virus-like particles. The supernatant wasremoved and the resulting pellet was resuspended in 3 ml PBS buffer (136mM NaCl, 2.7 mM KCl, 8.1 mM Na₂ HPO₄, 1.5 mM KH₂ PO₄). The resuspendedpellet was applied to a 15-45% continuous sucrose density gradient andcentrifuged for 90 minutes at 25,000 rpm in a SW28 rotor. Fractions werecollected dropwise. Samples from each sucrose gradient fraction weresubjected to immunoprecipitation analysis using macaque anti-SIVantiserum or mouse monoclonal anti-PRV gIII (M7) as described in Example4. The immunoprecipitates were analyzed by SDS-PAGE and the proteinbands visualized by scintillation autofluorography (Bonner and Laskey,European Journal of Biochem., 46:83-88 (1974)). SIV-specific proteinbands, including processed gag polypeptides, reverse transcriptase andendonuclease, co-sedimented in the gradient at a density expected forSIV particles. These results demonstrate that the pelleted materialcontains retrovirus-like particles, rather than simple aggregates ofretroviral polypeptides. The fractions were also analyzed for thepresence of PRV gIII. The results showed that the sucrose gradientfractions containing peak concentrations of gag-pol antigens alsocontained peak concentrations of the gIII antigen. These resultsstrongly suggested that the recombinant vaccinia-produced gIII, gag andpol proteins self-assemble into hybrid retrovirus-like particles.

EXAMPLE 7 Construction of a Divalent Vaccinia Recombinant Expressing SIVgag-pol and Equine Herpesvirus-1 (EHV-1) gB Genes Under the Control ofVaccinia Promoters

It is possible to produce hybrid viral particles from a singlerecombinant virus that expresses both the capsid polypeptides and aviral glycoprotein of interest. As an example, a recombinant vacciniavirus that contains the SIV gag-pol genes inserted at the HindIII Mregion of the genome (vAbT394) can be used as the parent for insertionof an envelope glycoprotein gene inserted in the thymidine kinase (TK)gene (in the HindIII J region of the genome) by in vivo recombinationwith an appropriate IVR vector. One IVR vector suitable for this purposeis pAbT817, the construction of which is described in PCT ApplicationNo. WO90/01546, published Feb. 22, 1990, the teachings of which areincorporated herein by reference. pAbT817 contains the equineherpesvirus-1 (EHV-1) glycoprotein B (gB) gene, under the control of thevaccinia 40K promoter, the vaccinia TK gene for directing recombinationin vaccinia, the E. coli lacZ gene under the control of the vacciniaBamF promoter for selection of recombinants and a bacterial replicon andampicillin-resistance gene for growth and selection in E. coli.

To generate a recombinant virus that co-expresses EHV-1 gB and SIVgag-pol, the IVR vector pAbT817 can be transfected into TK⁻ host cells(Hul42TK⁻) which have been infected with vAbT394. The desiredrecombinant, which will be TK⁻ due to the insertion of foreign DNA intothe vaccinia TK gene, can be selected using bromodeoxyuridine (BUdR),which is lethal for TK⁺ virus but allows recombinant TK⁻ virus to grow.In addition, the recombinant virus will contain the E. coli lacZ geneand express β-galactosidase. Thus, the recombinant virus can also beidentified by its ability to form blue plaques in the presence ofBluogal™.

The formation of capsids in cells infected with this recombinant viruscan be demonstrated essentially as described in the preceding examples.After infecting cells with the recombinant expressing both SIV gag-poland EHV-1 gB proteins, the culture medium can be analyzed bysedimentation, immunoprecipitation and PAGE methods described herein todemonstrate the production of virus-like particles containing the EHV-1gG glycoprotein and SIV capsid proteins.

EXAMPLE 8 Construction of a Recombinant Plasmid Vector Containing the gDGene of Herpes Simplex Virus Type 2 (HSV-2) (FIG. 3)

This Example illustrates the construction of recombinant plasmid vectorcontaining the HSV-2 gD gene (gD2) for insertion into vaccinia virus.

Plasmid p322gD-2, which was obtained from Vickie Landolfi(Lederle-Praxis Biologicals, Pearl River, N.Y.) was digested with SpeIand PstI and treated with Klenow fragment of DNA polymerase I. Theresulting 1400 bp fragment containing the gD2 gene was ligated toplasmid vector pAbT4587 (see, Example 2 above) which had been digestedwith SmaI and treated with calf intestinal phosphate, yielding plasmidpAbT1527. pAbT1527 contains the gD2 gene under the control of thevaccinia virus 40K promoter.

EXAMPLE 9 Construction of Recombinant Vaccinia Virus Containing the gDGene of HSV-2

To insert the gD2 gene into the vaccinia virus genome at the HindIII Msite of vaccinia virus, BSC-40 cells were infected with vAbT33,transfected with pAbT1527 and the recombinant virus selected andpurified by the scheme described in Example 3a. This generated vacciniarecombinant vAbT509. To confirm the presence of the gD2 gene in therecombinant viral genome, DNA was extracted from vAbT509-infected cellsand analyzed by restriction enzyme digestion with Southernhybridization, as described in Example 3c, using radiolabeled probescorresponding to the gD2 gene.

EXAMPLE 10 Immunoprecipitation of gD2 Antigen from Cells Infected withRecombinant Vaccinia Virus

Immunoprecipitation analysis was carried out as described in Example 4,using vAbT509 and an anti-gD2 monoclonal antibody, designated DL6,obtained from Vickie Landolfi (Lederle-Praxis Biologicals, Pearl River,N.Y.). The results confirmed production of gD2 antigen in cells infectedwith vAbT509.

EXAMPLE 11 Detection of Hybrid Retroviral Particles Produced byCoinfection with Vaccinia Recombinants vAbT509 and vAbT394

To demonstrate that coinfection of mammalian cells with vAbT394 andvAbT509 (gD2) results in the production of hybrid retroviral-likeparticles containing SIV core proteins and HSV gD2 envelopeglycoprotein, the following experiment was performed. Confluent BSC-40cells were infected at a multiplicity of infection of 5 pfu/cell witheither vAbT509 alone vAbT394 alone or vAbT509 and vAbT394 together. Theculture media were harvested at 20 hours post-infection, and clarifiedby two ten-minute centrifugations at 3,000 rpm. The particulate materialin each sample was then harvested by centrifugation at 120,000 g for 90minutes in an SW28.1 rotor. The pelleted material from each sample wasresuspended in 1.0 ml 10% glycerol in 10 mM Tris-HCl pH 7.2 andcentrifuged at 120,000 g for 90 minutes in an SW28.1 rotor through a15-45% linear sucrose gradient layered onto a 1.0 ml 60% sucrosecushion. The sucrose gradients were fractionated dropwise through thebottom of the tubes. The fractions were then chloroform/methanolprecipitated and the samples were subjected to electrophoresis on a 12%SDS-polyacrylamide gel. The separated proteins were elctrophoreticallytransferred to Millipore filters and the proteins reacted with eitherthe gD2-specific monoclonal antibody DL6 or with macaque anti-SIV serum.The filter-bound antigen/antibody complexes were visualized by reactionwith a secondary chemiluminescent antibody.

After sedimentation through the sucrose gradient, the gD2 in the pelletfraction from cells infected with vaccinia recombinant vAbT509 migratednear the top of the gradient; this glycoprotein is most likelyassociated with membrane fragments pelleted by the ultracentrifugationof the clarified medium. By contrast, a large proportion of the SIVpolypeptides contained in the pellet fraction from cells infected withvAbT394 were located in gradient fractions corresponding to the expecteddensity for lentivirus-like particles.

In contrast to the results obtained in the single infection, themajority of the gD2 contained in the pellet material from cellsco-infected with vAbT509 and vAbT394 co-sedimented with the SIVpolypeptides in gradient fractions corresponding to the density ofSIV-like particles. These results indicate that pseudotyped virus-likeparticles, comprising SIV core proteins and HSV gD2 glycoprotein, weregenerated in cells co-infected with recombinant vaccinia virusesexpressing these polypeptides.

EXAMPLE 12 Immunogenicity of gD2/SIV Virus-like Particles

A pseudotyped virus-like particles (VLP-gD2) preparation was preparedfrom supernatant media from 5 roller bottles of BSC-40 cell cultures(5×10⁸ cells) co-infected for 18 hours with vAbT394 and vAbT509 at amultiplicity of infection of 3 pfu/cell of each virus. The supernatantmedium was clarified by centrifugation two times at 3,000 rpm for 10minutes. The clarified supernatant was layered on top of a 25% sucrosecushion and centrifuged 90 minutes at 120,000 g in an SW28 rotor. Thesediments were resuspended in 500 ul of PBS and treated with 0.8%formalin at 40° C. overnight. A 5 ul sample of this material wasblind-passaged two times on BSC-40 cells without any visible signs ofinfection arising in the cell cultures. Six week old mice in groups of 5were immunized with 250 ul of material by either the intramuscular (IM)or the subcutaneous (SC) routes. The immunogen (VLP-gD2) preparationused for IM immunization was aluminum phosphate precipitated whereas thematerial used for SC immunization was not aluminum phosphateprecipitated. Three weeks later the mice were immunized again with thesame amount of material, treated in the same way and given by the sameroute as for the primary immunization. Thus, each mouse was immunizedwith a total of 1/10 the material generated in the 5 roller bottlestarting cell culture. Another 5 mice were immunized with 1×10⁷ pfu ofvAbT509 first by tail scarification (TS) and three weeks later byintranasal (IN) instillation. All mice were bled 2 weeks after thesecond immunization. Anti-vaccinia and anti-gD2 immune responses weredetermined by ELISA. The ELISA plates were coated with either a vacciniacell lysate or an HSV gD2 antigen purified from HSV-2 infected cells.Titer is defined as the reciprocal of the dilution which achieves 50% ofthe maximum value for the positive control (mouse anti-vaccinia sera ormonoclonal anti-gD2 DL6). Results are shown in Table 3.

                  TABLE 3    ______________________________________    Immunogenicity of Pseudotyped Virus-like Particles                   Anti-vac Titer                            Anti-gD2 Titer    Antigen    Route     of 5 mice  of 5 mice    ______________________________________    None       --        <10        <10    live vAbT509               TS, IN    480        480                         480        480                         640        1280                         960        1280                         1280       1280    VLP-gD2    SC, SC    <10        10                         <10        10                         <10        80                         <10        120                         <10        160    VLP-gD2    IM, IM    <10        20                         <10        80                         <10        120                         <10        120                         <10        1920    ______________________________________

Mice immunized with 1×10⁷ pfu of live recombinant vAbT509 first by tailscarification and three weeks later by intranasal instillation generatedantibodies against vaccinia antigens and the HSV gD2 antigen. Incontrast, mice immunized by the subcutaneous or intramuscular routeswith gD2/SIV pseudovirions developed antibodies against the gD2glycoprotein but the antibody response against vaccinia was severalorders of magnitude lower than in mice immunized with live vaccinia.Thus, both the gD2 glycoprotein expressed by the recombinant vacciniavirus during infection of mice with vAbT509 and gD2 glycoproteinrecovered in the pellet fraction after co-infection with cells withvAbT394 and vAbT509 elicit antibodies that recognize the purified gD2glycoprotein.

Plasmid Deposits

The plasmids pAbT4660 and pAbT4602 were placed on deposit, underprovisions of the Budapest Treaty, at the American Type CultureCollection (ATCC) in Rockville, Md. on Aug. 8, 1990, 1990. The plasmidshave been assigned ATCC Accession Nos. 40866 and 40865, respectively.

Plasmid pAbT1527 was deposited at the ATCC, under the provisions of theBudapest Treaty, on Aug. 9, 1991, and received Accession No. 75057.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims:

I claim:
 1. Plasmid DNA vector pAbT4602, pAbT4660 or pAbT1527 havingATCC designation numbers 40865, 40866, and 75057, respectively.
 2. A DNAdonor vector for insertion of DNA encoding a viral capsid polypeptideand an envelope glycoprotein from a different virus specie that selfassemble into a replication-defective, hybrid virus particle, into arecombinant DNA viral vector by in vivo recombination, comprising:a) aprokaryotic origin of replication so that the vector can be amplified ina prokaryotic host; b) a gene encoding a marker which allows selectionof prokaryotic host cells that contain the vector; c) a DNA sequenceencoding a viral capsid polypeptide and at least one other DNA sequencefrom a different virus encoding viral envelope glycoproteins thatself-assemble into a replication-defective, hybrid virus particle, eachDNA sequence located adjacent to a transcriptional promoter; and d) DNAsequence homologous to the region of the DNA viral genome where the DNAsequences will be inserted flanking the construct of element c.
 3. Amethod of producing a self-assembled replication-defective, hybrid virusparticle, comprising infecting a eukaryotic cell with a DNA viral vectorwhich coexpresses in eukaryotic cells, a heterologous gene encoding aviral capsid polypeptide and at least one gene encoding a viral envelopeglycoprotein from a different virus than the viral capsid, wherein theencoded capsid polypeptide and envelope glycoprotein self assemble intothe replication-defective, hybrid virus particle.
 4. A method ofproducing a self-assembled replication-defective, hybrid virus particle,comprising coinfecting a eukaryotic cell with at least two DNA viralvectors of the same virus specie, a first vector expressing aheterologous gene encoding a capsid polypeptide and a second vectorexpressing a gene encoding a viral envelope glycoprotein, wherein theencoded capsid polypeptide and envelope glycoprotein self assemble intothe hybrid virus particle.
 5. The method of claim 4, wherein at leastone of the genes encoding viral envelope glycoprotein or the geneencoding the viral capsid polypeptide is operably linked to an induciblepromoter.
 6. The method of claim 5, wherein the vector containing theinducible promoter is used to transform a cell.
 7. The method of claim5, wherein both the gene encoding the capsid polypeptide and the geneencoding the viral envelope glycoprotein are under the control of aninducible promoter.